Proper transgene expression is essential to achieve therapeutic effect in many gene therapy strategies. Current systems for regulating transgene expression typically require co-delivery of a cassette expressing a trans-activator protein adding significantly to the payload required of the chosen gene transfer vector. The concern of payload is exacerbated for adeno-associated virus (AAV) which has a packaging limit of less than 5 kb. To avoid the drawback, we are exploring a novel regulation system based on alternative splicing. Alternative splicing is the inclusion or skipping of an alternative exon in between two exons that are constitutively included during the process of pre-mRNA splicing. The inclusion of an alternative exon will result in synthesis of a truncated protein in our system. Other studies have shown that inclusion of the alternative exon could be blocked by anti-sense oligonucleotides (AON) complementary to an essential element of alternative splicing. Thus, skipping of the alternative exon is forced to take place resulting in synthesis of a functional full length protein. Controlling transgene expression mediated by adeno-associated virus (AAV) is confronted by a challenge of using minimal elements. Our long-term goal is to develop novel delivery systems that exploit the advantages of AAV mediated persistent gene transfer with the ability to control the expression of the therapeutic genes being delivered. The hypothesis of our proposed studies is that by incorporating an optimized alternative splicing mechanism into the AAV delivery system, both long- term and controllable transgene expression could be achieved in vivo.
The Specific Aims are: 1) Optimize a small intron capable of alternative splicing, and 2) Incorporate optimized introns capable of alternative splicing into AAV vector to control transgene expression over time in an animal model. The experiments described in this proposal would allow us to develop and optimize the alternative splicing as a general means of controlling transgene expression mediated by AAV. Such a mechanism could also be used by other gene transfer vectors including lentivirus and other retroviruses, adenovirus, herpes virus as well as artificial chromosome. Proper transgene expression is essential to achieve therapeutic effect in many gene therapy strategies. Current systems typically have a demanding payload required of the chosen gene transfer vector. This concern is exacerbated for AAV which has a packaging limit of less than 5 kb. To overcome the current drawback, we are proposing a novel regulation system for controlling the expression of the therapeutic genes being delivered. ? ? ?
